The success of Mycobacterium tuberculosis, and other pathogenic mycobacteria species, lies in their ability to modulate their phagosome and prevent it from differentiating into an acidic, hydrolytically-competent compartment. The consensus in the field is that the bacterium arrests the normal maturation process leading to retention of the vacuole within the cell's recycling/sorting endosomal system. Although many studies detail the presence or absence of host molecules implicated in control of membrane fusion, the mechanism by which the bacterium modulates the phagosome remain to be determined. This proposal describes an integrated series of projects that will address this issue, place it in the context of the normal maturation process for phagosomes, and determine the consquences to the bacterium if it is delivered to the lysosome. The proposal addresses the following three aims: 1. Analysis of the lipid constituents of the phagosome membrane during maturation of IgG-bead and M. tuberculosis-containing phagosomes. The phosphorylation status of several membrane lipids changes during phagosome biogenesis and these alterations drive the association with the membrane fusion machinery in the cell. We intend to map these changes and correlate them with phenotypic differences in the pathogen-containing compartment. 2. Isolation and characterization of M. tuberculosis mutants defective in modulation of the phagosome. We have developed a genetic screen for such mutants and are currently characterizing the genetic defects that prevent modulation of the phagosome. These mutants should provide the tools that we need to determine how the bacterium regulates its phagosome. 3. Identification of lysosomal constituents that are bactericidal to M. tuberculosis. We have found that isolated lysosomes kill M. tuberculosis when added to bacterial cultures. Preliminary characterization of this fraction suggests that the activity co-purifies with cathepsins S and B. We intend to identify the active component and characterize the mechanism that leads to bacterial death.